Exploring strategies to optimise outcomes in hepatitis-associated aplastic anaemia patients following haematopoietic stem cell transplantation

This study aimed to assess haematopoietic stem cell transplantation (HSCT) safety and efficacy while exploring strategies for optimising outcomes in patients with hepatitis-associated aplastic anaemia (HAAA). We retrospectively reviewed 35 HAAA patients who underwent HSCT at a large Chinese blood disease hospital between 2008 and 2022. HAAA patients receiving HSCT typically presented with severe (28.6%) and very severe (65.7%) AA. Male patients predominated (68.6%), with a median onset age of 23 years (range, 9–44). Haploidentical donor-HSCT and matched sibling donor-HSCT were in comparable proportions. The 5-year overall survival (OS) rate was 74.0%, with cumulative incidences of grade II–IV acute and chronic graft-versus-host disease (GVHD) at 37.1% and 22.4%, respectively. A diagnosis-to-HSCT interval ≥ 75 days, acute GVHD, and post-HSCT liver events (e.g., hepatic GVHD and a three-fold increase in aminotransferase or bilirubin) significantly worsened 5-year OS. In the multivariate models, recipients with sex-matched grafts had better OS, and those with younger male donors had a lower incidence of II–IV aGVHD. Higher HLA matching degree (HLA > = 7/10) was an independent prognostic factor associated with better OS and GFFS. A diagnosis-to-HSCT interval ≥ 75 days was predictive of post-transplant liver events in HAAA patients. In conclusion, HSCT was a safe and effective treatment for HAAA. Early transplantation, careful donor selection and improving post-transplant liver events were crucial to optimise outcomes.


Patients
We conducted a retrospective analysis of 35 consecutive patients who underwent HSCT for HAAA between September 2008 and August 2022 at a 766-bed tertiary blood disease hospital in Tianjin, China.AA was diagnosed according to the Camitta criteria and patients with paroxysmal nocturnal haemoglobinuria (PNH) clones were included in the study, while those with hereditary bone marrow failure syndromes and myelodysplastic syndrome were excluded 7 .HAAA was defined as a specific subtype of AA, characterized by onset of pancytopenia with a hypoplastic bone marrow that traditionally occurs within 6 months of an elevation in serum aminotransferases 2,8 .Serological and virological testing confirmed negative hepatitis A, B, and C status.The study was approved by the Ethics Committee of the Institute of Haematology and Blood Diseases Hospital, and informed written consent was obtained from all patients or their guardians according to the Declaration of Helsinki.

Infection prevention strategies
Before transplantation, all patients received Paediatric Compound Sulfamethoxazole Tablets (1 g, twice daily for one week) to prevent Pneumocystis jirovecii pneumonia, alongside ganciclovir intravenously at a dosage of 10 mg/kg/day for 1 week to prevent CMV infection.For those without a pre-transplant invasive fungal disease (IFD) diagnosis, fluconazole was administered as a prophylactic treatment up to 3 months posttransplantation.Those diagnosed with IFD before transplantation were treated individually with itraconazole, voriconazole, micafungin, or caspofungin, depending on their specific pre-transplant conditions.Additionally, routine prophylactic treatment included antibiotics such as compound sulfamethoxazole and albendazole before transplantation.

Definitions
Neutrophil engraftment was defined as the first occurrence of three consecutive days with an ANC ≥ 0.5 × 10 9 /L without G-CSF, while platelet engraftment was defined as the day when the platelet count reached ≥ 20 × 10 9 /L without transfusion for a week.Using short tandem repeat PCR, cells > 95% indicate complete donor chimerism, reflecting total donor-derived haematopoiesis.Donor cells between 5.0 and 95% suggest mixed chimerism.Levels under 5% represent recipient-exclusive haematopoiesis, indicating absent donor chimerism 9 .Primary graft failure (GF) was defined as not achieving neutrophil engraftment by day + 28, while patients with initial engraftment who later experienced recurrent pancytopenia with obviously hypocellular BM were considered to have secondary GF.CMV viremia was characterized as a positive detection in blood samples, identified through reverse transcriptase PCR, with a threshold of 1 × 10 3 copies/mL.Post-transplant liver events were defined as veno-occlusive disease (VOD), viral hepatitis, hepatic GVHD, and a three-fold increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST) or total bilirubin (TBIL) within 6 months post-HSCT.aGVHD was identified through its clinical features and graded according to the MAGIC criteria 10 .For cGVHD, the diagnosis and grading were based on the 2014 National Institutes of Health consensus 11 .TRM was defined as the incidence of death without disease progression.Death from non-transplant causes was considered a competing risk for TRM, and death from any cause was a competing risk for engraftment and GVHD.OS was defined as the time from HSCT to death from any cause or last follow-up.FFS was defined as survival with treatment response, where death, GF, and relapse were considered treatment failures.GFFS was defined as survival without grade III-IV acute GVHD, moderate to severe chronic GVHD, and treatment failures as described above.The normal range used for the primary analysis were as follows: 0-50 U/L for ALT, 0-50 U/L for AST, 5-21 μmol/L for TBIL, 0-3.4 μmol/L for direct bilirubin (DBIL), 23.9-336.2ng/mL for serum ferritin, 56-86% for the proportion of CD3+ T cells, 33-58% for the proportion of CD4+ T cells, 13-39% for the proportion of CD8+ T cells, 5-22% for the proportion of CD19+ B cells, and 5-26% for the proportion of CD3−CD16/CD56+ NK cells to lymphocytes in PB.

Patient consent
We have obtained written informed consent from the patient or patient's parent/guardian.

Basic characteristics and transplantation details
Table 1 summarizes the clinical characteristics of 35 patients who underwent allo-HSCT for HAAA over the past 15 years, with a median follow-up time of 1156 days (range, 130-5099) for survivors.Our study revealed that HAAA patients who received HSCT were more male (n = 24, 68.6%), with a median onset age of 23 years (range, 9-44), and only one patient was over 40 years old.The majority of HAAA cases were VSAA (n = 23, 65.7%) or SAA (n = 10, 28.6%).Mucocutaneous hemorrhage was the primary symptom in 60.0% of HAAA patients, followed by infections with or without fever (37.1%), while 17.1% of patients presented with fatigue only.The median time from hepatitis to diagnosis was 60 days (range, 0-210).For patients with available medical information at the onset of hepatitis, over half had AST > 10 × upper limit of normal (ULN) (14/23), ALT > 20 × ULN (17/25), TBIL > 5 × ULN (11/19), and DBIL > 10 × ULN (13/17).Through meticulous serologic and virologic testing, we established that patients in our study were not affected by hepatitis A, B, or C. Additionally, we rigorously evaluated and subsequently excluded the possibility of drug-induced hepatitis in all cases.The majority of patients had high serum ferroprotein levels at HAAA diagnosis, with a median value of 567.85 (interquartile range [IQR], 299.43-905.40)ng/mL.Only 9.45% (n = 3) of the 32 evaluable patients had PNH clones.
Moreover, lymphocyte proportions are displayed in Fig. 1.Of the 23 patients with pre-treatment lymphocyte subset data, more than half of the HAAA patients (56.5%, n = 13) had a CD4 cell proportion below the normal level prior to treatment, and 1/3 of the patients (34.8%, n = 8) had a CD3−CD16/CD56+ T cell proportion below normal, suggesting that the PB lymphocyte imbalance plays a central role in the immune-mediated pathogenesis of HAAA.
Additionally, we found that the median time from diagnosis to HSCT in HAAA patients was 75 days (range, 34-412) (Table 2).Prior to HSCT, two patients received immunosuppressive therapy based on ATG.None of the HAAA patients underwent matched unrelated donor (MUD)-HSCT, while haploidentical donor (HID)-HSCT (n = 18) and MSD-HSCT (n = 17) were used in similar proportions.The degree of donor-recipient blood type and sex matching was unevenly distributed.All patients had a haematopoietic cell transplantation specific comorbidity index of 0-1.The vast majority of HAAA patients received GVHD prophylaxis with CsA + MTX ± MMF (84.4%) and conditioning regimen with Cy + Flu + ATG (82.9%).PB was the primary source of stem cells for HAAA patients (82.9%, n = 29).The median mononuclear cell count (MNC) was 10.00 × 10 8 / kg of recipient weight, and the median CD34+ cell count was 2.92 × 10 6 /kg of recipient weight, respectively.

Liver events
Our study focused on post-transplant liver events in HAAA patients.We found that 40% of the patients (14 in total) experienced hepatic events.We found that 40% of the patients (14 in total) experienced hepatic events after transplantation.Among these, eight patients developed hepatic GVHD, while the remaining 6 experienced significant enzyme elevation due to various other causes.None suffered from VOD and viral hepatitis.The 5-year OS rate was significantly lower for patients who developed post-HSCT liver events (51.4% [95% CI: 26.9-82.8%]vs. 90.2%[95% CI: 83.0-100.0%],P = 0.035) (Fig. 3A).

Discussion
HAAA is a relatively rare condition, accounting for approximately 3-12% of newly diagnosed acquired AA cases 2,3,12 .The mean interval between onset of hepatitis and first indication of AA was 2 months, which was consistent with previous studies [13][14][15] .Markedly elevated aminotransferases and conjugated hyperbilirubinemia were observed at diagnosis of hepatitis preceding pancytopenia.We investigated various aspects of post-transplant outcomes in patients with HAAA.In our research on HAAA patients, a substantial 65.7% had very severe aplastic anemia.They exhibited low initial platelet counts www.nature.com/scientificreports/(median 10 × 10 9 /L) and marked anemia (HGB, median 77 g/L).This led to a high transfusion requirement, contributing to notably higher ferritin levels in these patients, indicative of potential iron overload issues.The majority of patients (94.3%) opted for transplantation over ATG-based immunosuppressive therapy.The 2010 report from the EBMT aplastic anaemia working party 2 demonstrated a 10-year actuarial survival rate of 70% after transplantation, with most HAAA patients receiving MSD-HSCT.More recently, only one study conducted by the Japan Society for Haematopoietic Cell Transplantation 16 reported a 5-year overall survival (OS) rate of 86.0% and a 100-day cumulative incidence of grade II-IV acute graft-versus-host disease (aGVHD) at 11.1% among HAAA patients who received HSCT from HLA-identical donors.In our published article 4 , we conducted a comparative analysis, matching 30 patients who underwent HAAA procedures at our center with patients without HAAA.The findings showed that estimated 5-year OS rate (75.8% vs. 86.5%,P = 0.409) and FFS rate (74.0% vs. 83.2%,P = 0.485) after HSCT were slightly lower but not statistically significantly different from those in the non-HAAA group.All HAAA patients achieved neutrophil engraftment and the 30-day cumulative incidence of platelet engraftment was 88.6%.Overall, HAAA patients had satisfactory outcomes after transplantation.
In this study, we first analysed the risk factors influencing survival, aGVHD, and early CMV viremia in patients with HAAA.Our findings emphasized the importance of early referral, with a 75-day cut-off providing optimal separation of patients receiving early and late HSCT, resulting in different OS rates (100% vs. 46.6%,P = 0.001).Our cut-off value was significantly shorter than the waiting period from diagnosis to transplantation in previous studies, which affects survival in idiopathic AA (> 12 months, HR: 2.18, P = 0.027) 17 and all acquired AA (> 3 months, RR: 1.44, P = 0.031) 18 .www.nature.com/scientificreports/ We placed great importance on post-transplant liver events within 6 months for HAAA patients.A study from China 6 identified two liver events after HSCT in 15 HAAA patients who underwent HID-HSCT, and a study from Japan 16 identified one case of VOD in 37 adult HAAA patients who underwent MSD or MUD-HSCT.In our study, 40% of patients (14 in total) experienced hepatic events post-transplantation.Among them, 8 were diagnosed with hepatic GVHD, while 6 others showed significant liver enzyme elevations from varied causes.Two patients improved after modifying potentially hepatotoxic drugs and receiving hepatoprotective therapy, indicating drug-related liver issues.One patient, despite initial improvement after stopping tigecycline and receiving hepatoprotective therapy, ultimately succumbed to complex complications, leaving the cause of liver dysfunction unresolved.Another patient's liver condition worsened due to CMV and EBV viremia, ultimately proving fatal.The remaining two patients showed improvement with hepatoprotective therapy alone, making the precise cause of their post-transplant hepatitis unclear.This data highlights the multifaceted nature of posttransplant liver events, encompassing factors like drug toxicity, viral infections, and GVHD.This study is the first to demonstrate that post-transplant liver events, which have not been identified as a risk factor in patients transplanted for idiopathic AA, have an adverse impact on the survival of patients with HAAA.Our findings emphasize the importance of the timely intervention in mitigating post-transplant liver events.
Our study also confirmed that HLA locus matching < 7/10 was an independent risk factor that significantly worsened OS (HR: 8.2, 95% CI: 2.6-19.7,P = 0.006) and GFFS (HR: 4.4, 95% CI: 1.3-15.5,P = 0.019) in HAAA patients.In addition, many studies [19][20][21] have confirmed that AA patients who receive HID, MSD, or MUD-HSCT have similar 3-year OS and FFS rates, but the HID group still has a higher incidence of GVHD and early CMV viremia.We also found that HAAA patients were more likely to experience 100-day CMV viremia after HID-HSCT (64.1% vs. 17.6%,P = 0.010).The gender and age of the donors also affect the outcome of the transplant.
As with recipients with AA 22 , aGVHD was more common and survival was marginally worse in the older donor group with HAAA.Additionally, a study involving 1481 patients who underwent HSCT for AA from 28 countries 23 reported a significantly higher 5-year OS in the donor-patient sex-matched group (68% vs. 60%, P = 0.001), and male patients with female donors were at an increased risk of severe GVHD (RR: 1.33, P = 0.03).
In our adjusted multivariate models, we also observed better survival in HAAA recipients with sex-matched grafts and a lower incidence of aGVHD in recipients with male donors.Therefore, a younger male donor may predict a better post-transplant outcome for HAAA patients, and transplantation from donors with a higher degree of HLA matching may result in improved survival rates.Pursuing higher doses of certain cells in allografts may be counterproductive due to poorer survival.The optimal CD34+ cell count is uncertain and varies by graft type.Several studies [24][25][26] supported that higher CD34+ progenitor doses improve OS, but others 27,28 argued that excessively high CD34 cell counts may lead to elevated TRM (> 5 × 10 6 /kg) and extensive cGVHD morbidity (> 8 × 10 6 /kg).In addition, the correlation between CD3+, CD4+ and CD8+ doses in the graft and post-transplant outcomes remains controversial 25,29,30 .We found that in HAAA patients, higher doses of CD34+ (> 4.5 × 10 6 /kg), CD4+ (> 125.0 × 10 6 /kg) and CD8+ (> 52.0 × 10 6 /kg) resulted in poorer 5-year OS, and higher MNC (> 12.5 × 10 8 /kg) and CD3+ (> 115.0 × 10 6 /kg) doses were also associated with poorer 5-year GFFS.Table 4. Univariate and multivariate analyses of adverse factors associated with II-IV aGVHD and CMV viremia.A P value in italics and bold means < 0.1, followed by * means < 0.05, followed by # means that this variable was included in the multifactorial analysis.aGVHD acute graft-versus-host disease, ATG anti-thymocyte globulin, CsA cyclosporin, Bu busulfan, CMV cytomegalovirus, Cy cyclophosphamide, Flu fludarabine, HID haploidentical donor, MSD matched sibling donor, PLT platelet.

Figure 1 .
Figure 1.Proportion of peripheral blood lymphocyte subsets in HAAA patients before treatment.The median proportion of each lymphocyte subset to lymphocytes is shown with a solid black line, and the normal reference range for lymphocyte subsets is represented with a dashed black line.

Table 3 .
Univariate and multivariate analyses of adverse factors associated with OS and GFFS.